1. Field of Invention
The field of this invention relates to antigenic stimulation of specific immune responses. Specifically, the invention relates to identification of B cell superantigens and their use as adjuvants and/or carrier proteins to enhance a specific immune response to bacterial or viral pathogens having polysaccharide or glycoprotein components in their cell walls, cell membranes, capsules or envelopes. More particularly, it relates to a method of enhancing the immune response by administration of said polysaccharide or glycoprotein with a B cell superantigen either concomitantly or in a chemically conjugated form.
2. History of the Prior Art
For a complete understanding of the invention, a brief summary of the role of clonal development of B cells in immune responses is helpful.
In the germ-line cells, there are three sets of germ-line genes involved in immunoglobulin coding: one set codes for the heavy chains, and the other two code for two types of light chain designated by the Greek letters kappa (.kappa.) and lambda (.lambda.), which differ significantly in the amino acid sequence of their constant domains. Immunoglobulins are composed of heavy and light chain heterodimers, which contribute to the conventional antigen binding site in the Fab portion. B cells themselves are lymphocytes which have immunoglobins on their cell surface which serve as receptors for antigens. Binding of antigen, in most instances together with additional signals from T cells, causes the original B cell to proliferate, forming clones. The expanded clonal population differentiates into memory cells and plasma cells, the latter of which synthesize and secrete antibodies which generally will have identical binding sites for the antigen which triggered the B cell activation. Polyclonal activation occurs when certain antigens (known to include mitogens and the Epstein-Barr virus) stimulate clonal expansion regardless of the antigen specificity of the B cells involved.
The human antibody repertoire is responsible for the acquisition of a dynamic and responsive immune defense system, but dysregulation within the B cell compartment can result in a wide spectrum of clinical conditions, including inappropriate B cell clonal expansions (e.g., B cell neoplasms), inadequate immune defense from infection, or autoimmune disease. During fetal development, antibody specificities are acquired slowly in a developmentally ordered fashion.
Referring for purposes of illustration to humans, the V, J, and D genes code in human cells for the variable regions of the antibody molecule and the C genes code for the constant regions. Each of the three germ-line sets contain from two to at least 300 alternative V genes, together with a small number of alternative J genes; the heavy-chain set also has alternative D genes. Any of these genes can contribute in the variable regions of antibody molecules. Each set has from one to five alternative C genes coding for different constant regions.
As a germ-line cell differentiates into a mature but "naive" B cell (i.e. one that is reactive to but has not yet encountered its matching antigen), somatic recombination of the germ-line genes takes place. In each cell, one of the V genes from each of the three germ-line sets is "selected" by an unknovin molecular process, together with one of the adjacent J genes (and in heavy chains, also with one of the D genes). These selected genes are brought together in the genome when the intervening DNA is excised. The basis of diversity in the antigen-recognition structure of an antibody molecule rests initially on this recombination event, since different genes are recombined in different B cells.
Although the first source of variation in the structure of the antibody molecule is brought about by somatic recombination of alternative V, D and J genes, further diversity in the amino acid sequence of the variable domains results from variable recombinations; i.e., slight variations in the exact location of the "cutting points" as first the germ-line DNA and later the mRNA transcripts are cut and spliced. Both these sources of variation occur before contact with antigen.
More variation arises after contact with antigen. Single base changes ("somatic mutation") occur in the DNA of activated B cells, mainly during the process of memory-cell formation.
A consequence of somatic mutation is that some of the binding sites produced by the mutated DNA have a better affinity for the antigen, and some have a worse affinity. Somatic mutations occur mainly during clonal expansion and memory-B cell formation, so the memory B cells from a single clone end up with receptors (i.e. surface immunoglobulin) for the same antigen, but with a range of antigen affinities (see, re development of the human antibody repertoire generally, Davey, Immunology: A Foundation Text (1990) Chapter 4, sections 4.3-4.3.2).
The process of repertoire selection may be the result of long-term exposure to many exogenous and endogenous contentional ligands, but a dramatic skewing of the immune repertoire can also be induced by a single limited exposure to certain unconventional antigens. These antigens, of bacterial or viral origin, were first distinguished based on an ability to interact with a large proportion of T lymphocytes. In contrast to conventional antigens, which generally stimulate less than 0.01% of T cells, these superantigens can stimulate 5-25% of all T cells. In explanation, many superarntgens are recognized by most (or all) T cells that use a particular V.beta. family. Based on available data, superantigen reactivity is little affected by differences in V.beta. junctional sequences, or by the alpha chains that are co-expressed. Moreover, recent studies indicate that an alternative site, remote from conventional antigen binding sites, allows for superantigen recognition by a large proportion of the T cell pool, usually those having particular V.beta. elements.
Superantigens have been proposed to contribute to the shaping of the mature T cell repertoire by clonal selection and/or deletion. Individual inbred strains of mice have been shown to carry different murine Mammary Tumor Virus encoded endogenous superantigens, and each exhibits a distortion of the distribution of T cell receptor V.beta. expression. This is usually due to deletion of T cells with certain V.beta. families, although there may also be more subtle effects on positive selection. Similar acute fluctuations may also occur in patients with toxic shock syndrome and Kawasaki's disease, presumably due to a T cell superantigen. Several recent papers have also suggested that superantigen exposure of predisposed individuals may, at times, result in immunosuppression, the production of autoantibodies, the development of autoimmune disease, or the abolition of an autoimmune process (see also, re characteristics of T cell superantigens, Fraser, et al. (1992) J. Exp. Med 175:1131-1134, and Taub, (1992) Cell Immunol. 140:267-281).
With this background, interest in the possibility that B cell superantigens (hereafter sAg) may exist can be understood. Several candidate sAg's are suggested by recent reports. For example, human IgM, IgA and IgGF(ab').sub.2 that bind to bacterial membrane protein staphylococcal protein A (SpA) have been shown to include a family of V.sub.H genes which encode polypeptides belonging to the V.sub.H 3 protein subgroup (the largest human family) (Sasso, et al., J. Immunol. (1991) 147:1877-1883 and Sasso, et al., J. Immunol. (1989) 142:2778-2783). As described further herein, SpA binds to the Fab region of a large proportion of V.sub.H 3 restricted immunoglobins at an alternative binding site different from its known, F.sub.C .gamma. binding sites and in greater proportion than conventional antibody binding. Further, the Fab sites which bind SpA are found on antibodies with diverse specificities.
Protein F.sub.V, a sialoprotein released into the digestive tract during viral infection, has also been reported to interact with the Fab fragment of immunoglobins at an unconventional binding site believed to be in the V.sub.H domain (Bouvet, Scand. J. Immunol. (1991) 33:381-386). Further, based on reported binding by human Ig via the Fab region (in a setting in which prior immunization to create antibodies to conventional components is not required), known protein components of other microorganisms may also serve as B cell sAg.
The present invention includes a means of characterizing and identifying B cell sAg and using them to enhance production of V.sub.H, particularly V.sub.H 3, restricted antibodies. In particular, the sAg will be identified, purified and administered concomitantly with a polysaccharide or glycoprotein component from a bacterial or viral cell wall or capsule or, preferably, as a carrier for a conjugate vaccine to the bacteria or virus. B cell superantigens with specificity for other Variable (V) region gene families may also exist, and be useful in certain vaccines.
In the past, there has been no rationale for selection of carriers for conjugate vaccines except the experience with these proteins as immunogens themselves. In each case, diphtheria toxoid, tetanus toxoid and OMB (outer membrane protein of N. meningitides) the carriers were selected and/or used clinically to elicit immunity to the pathogen. As a result they were shown to be immunogenic and safe for human use. Prototype conjugate vaccines were then made and tested in animal models, usually rabbit or mice. The only possible exception is the present OMB conjugate, which uses a liposome delivery system (Donnely et al., J. lmmunol (1990) 145: 3071-3079).